title: Dynamin-related protein 1 regulates substrate oxidation in skeletal muscle by stabilizing cellular and mitochondrial calcium dynamics

status: Public on Sep 13 2021

city: Baton Rouge

first: John

middle: P

last: Kirwan

state: LA

address: 6400 Perkins Road

institute: Pennington Biomedical Research Center

summary: Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, inhibition of ATP-stimulated calcium flux, and impaired substrate oxidation stimulated by calcium levels. The insights obtained herein suggest that DRP1 regulates fatty acid oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases.

overall_design: Differentiated cells were lysed and total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was quantified using NanoDrop and normalized to 200 ng/μL in nuclease-free water. RNA integrity was assessed with Agilent Bioanalyzer 2100. Libraries were constructed and sequenced using Lexogen QuantSeq. Briefly, library generation was performed using an oligodT primer, and double-stranded cDNA was purified with magnetic beads. Libraries were amplified using PCR, and transcripts were forward sequenced at 75 bp using NextSeq 500 (Illumina). BlueBee software was used to analyze alignment and DESeq2 was used for differential expression analysis. n=5-6 per group

first: W

middle: T

last: King

first: C

last: Axelrod

first: J

first: Susan

last: Newman

first: Richard

last: Carmouche

first: Jasleen

last: Kaur

first: Sara

last: Webb

first: G

last: Davuluri


title: CD8+ TILs in NSCLC differentiate into TEMRA via a bifurcated trajectory, deciphering immunogenicity of tumor-antigens

status: Public on Sep 14 2021

city: Hwasun-eup, Hwasun-gun

first: Jae-Ho

last: Cho

state: Jeollanam-do

address: 264, Seoyang-ro

country: South Korea

institute: CHONNAM NATIONAL UNIVERSITY

type: Expression profiling by high throughput sequencing

summary: CD8+ tumor-infiltrating lymphocytes (TILs) comprise phenotypically and functionally heterogeneous subpopulations. Of these, effector memory CD45RA-expressing CD8+ T cells (Temra) have been discovered and characterized as the most terminally differentiated subset. However, their exact ontogeny and physiological importance in association with tumor progression remain poorly understood. We analyzed primary tumors and peripheral blood samples from 26 patients with non-small cell lung cancer and analyzed their phenotypes and functional characteristics using flow cytometry, RNA-sequencing, and bioinformatics. We found that tumor-infiltrating Temra (tilTemra) cells largely differ from peripheral blood Temra (pTemra), with distinct transcriptomes and functional properties. Notably, although majority of the pTemra was CD27-CD28- double-negative (DN), a large fraction of tilTemra population was CD27+CD28+ double-positive (DP), a characteristic of early-stage, less differentiated effector cells. Trajectory analysis revealed that CD8+ TILs undergo a divergent sequence of events for differentiation into either DP or DN tilTemra. Such a differentiation toward DP tilTemra relied on persistent expression of CD27 and CD28 and was associated with weak T cell receptor engagement. Thus, a higher proportion of DP Temra was correlated with lower immunogenicity of tumor antigens and consequently lower accumulation of CD8+ TILs. These data suggest a complex interplay between CD8+ T cells and tumors and define DP Temra as a unique subset of tumor-specific CD8+ TILs that are produced in patients with relatively low immunogenic cancer types, predicting immunogenicity of tumor antigens and CD8+ TIL counts, a reliable biomarker for successful cancer immunotherapy.

overall_design:
NGS data of tumor infiltrating Temra (tilTemra) and peripheral blood Temra (pTemra) from NSCLC patient and pTemra from healthy donor
3 independent experiments were carried out (1st: tilTemra_001, pTemra_001, pTemra_004; 2nd: tilTemra_002, pTemra_002, pTemra_005; 3rd: tilTemra_003, pTemra_003, pTemra_006) then analyzed together.
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Authors state "Raw data is unavailable due to patient privacy concerns".

first: Deok

middle: H

last: Yang

first: Sang

middle: Y

last: Song

first: Sung-Woo

last: Lee

first: He

last: Choi


title: NAD + Supplementation by Nicotinamide Riboside Alters Function of Murine Hematopoietic Stem Cells from Impaired Environments

city: Baltimore

first: Bongsoo

last: Park

state: Maryland

address: 251 Bayview Blvd

department: Translational Gerontology Branch

country: USA

institute: National Institute on Aging

summary: NAD+ supplementation has been shown to impart significant benefits in compromised settings, acting largely through improved mitochondrial function and DNA repair. Elevated NAD+ has been shown to improve the function of some adult stem cells, with implications that these changes could lead to sustained improvement of the tissue or system. We examined the effect of elevating NAD+ levels in models with reduced hematopoietic stem cell (HSC) potential (ATM-deficient and aged mice) and show that supplementation of NR, a NAD+ precursor, leads to improved lymphoid lineage potential and long-term cell-autonomous benefits. Transcriptional profiles from HSCs show changes driven by NR repress myeloid gene signatures, which may lead to the increased lymphoid output. Our work characterizes the improved lymphoid-potential from short-term NR treatment in functionally compromised HSCs and highlights potentially detrimental aspects of supplementing NAD+ to proficient systems.

overall_design: RNA sequencing was performed to compare the transcriptome between NR treated HSC and control HSC.

first: Le

last: Zong

first: Mayuri

last: Tanaka

first: Hagai

last: Yanai

first: Ferda

last: Thrhan

first: Deborah

middle: L

last: Croteau

first: Jane

last: Tian

first: Evandro

middle: F

last: Fang

first: Vilhelm

middle: A

last: Bohr

first: Isabel

last: Beerman


title: Characterizing the Human Menstrual Cycle from a gene co-expression perspective reveals the pregnant endometrium as a global transcriptionally derepressed state

status: Public on Sep 15 2021

city: Valencia

first: Patricia

last: Sebastian-Leon

state: Valencia

address: EDIFICIO BIOPOLO - LA FE, Avenida Fernando Abril Martorell, 106 - Torre A, Planta 1ª

department: Genomics and Systems Reproductive Medicine

country: Spain

institute: IVI-RMA Foundation

summary:
Background: a fundamental challenge for reproductive biology is to understand the menstrual cycle, specially the window of implantation, whose regulation and mechanistic remain incompletely understood in human reproductive physiology. Transcriptomics from a systems biology perspective provides a genomic framework to discover endometrial tissue specific regulation and behavior and its function in reproductive physiology.
Results: Here, we applied a transcriptomic analysis of 238 endometrial receptivity related genes using a customized Agilent platform. 507 human endometrial biopsies were analyzed among the menstrual cycle, especially in the expected window of implantation and divided in to transcriptomic profiles using machine learning predictors (SVM and KNN models). The clinical relevance of these profiles was tested defining the best transcriptomic profile for pregnancy and live birth.   Weighed Gene Co-expression Networks (WGCN) was applied is these clinically well-defined endometrial states to characterize the gene co-expression tissue specific regulation in humans for pregnancy in comparison with the other transcriptomic stages of menstrual cycle. For the first time, the menstrual cycle has been mapped as a percentage of positive and negative correlations between genes. Thanks to this approach, we have discovered the WOI as the fewest negatively correlated state with the highest proportion of positive correlations (Cochran´s Q = 14324.12, FDR < 2.2e-16) with an average of  84% of positive correlations in pregnant profile, while 53% in the two previous Proliferative (PF) and Early Pre-Receptive profiles and 67%  in the late secretory endometrium, just before menstruation renewal. Finally, this study provides a wealth of network data to the scientific community to stimulate hypothesis-driven single-molecule endometrial studies in the form of a user-friendly database called the Menstrual Cycle Gene Co-expression Network (website: www.menstrualcyclegcn.com).
Conclusions: this genomic research reveals for first time the pregnant endometrium as a global derepressed state in human menstrual cycle. This indicates the endometrium is generally a repressive tissue in human menstrual cycle with a relaxed WOI period where gene expression reaches the highest proportion of positive correlation, indicating that a decrease of inhibition may allow embryo implantation. Detailed molecular information about co-expression among menstrual cycle is also provided.

overall_design: Total 529 samples in six different menstrual cycle phases: PF, EPR, LPR, RR, LR, PS

first: Alejandro

last: Alemán

first: Almudena

last: Devesa-Peiro

first: Vicente

last: Arnau

first: Antonio

last: Pellicer

last: Diaz-Gimeno


title: Single-cell RNA sequencing for the identification of early-stage lung cancer biomarkers from circulating blood

city: Halifax

first: Paola

last: Marignani

state: Nova Scotia

address: 5850 College Street, Tupper Medical Building

department: Biochemistry & Molecular Biology

country: Canada

institute: Dalhousie University

summary: To develop biomarkers for diagnosis of early-stage lung adenocarcinoma (LUAD) patients, we set up single-cell RNA sequencing (scRNA-seq) pipeline using Fluidigm C1 systems. Through the scRNA-seq pipeline, we clustered 1,441 single cells from four non-small cell lung cancer (NSCLC) cell lines (A549, H460, H1299 and Calu3) into four groups based on differential gene expression and detected over 2,400 differentially expressed genes (DEGs) from a comparison of four clusters following dimension reduction. We validated fold change values of 120 DEGs selected from three cluster comparisons (top 20 up- and top 20 down-regulated genes per cluster comparison) using qRT-PCR approach. We performed further quantitative validation of chemokine genes (CXCL1 and CXCL2 with corresponding receptor, CXCR2) at mRNA level in tumour and tumour-adjacent normal lung tissues of LUAD patients (16 female and 19 male). Next, we profiled microRNAs epigenetically regulating expression of those chemokine genes in the tumour and normal tissues as well as blood (plasma) samples from corresponding LUAD patients. Finally, we identified relatively high copy number of miR-532-5p regulating those chemokine genes in patient plasma samples.

overall_design: We loaded 400 single cells per NSCLC cell lines (A549, H460, H1299 and Calu3) to chambers of high-throughput (HT) scRNA-seq integrated fluidics circuits (IFCs). We generated four hundreds of 3'-end enriched scRNA-seq libraries per NSCLC cell line using  in Fluidigm C1 systems (1,600 libraries in total). Those libraries were sequenced using illumina Nextseq 500 systems.

first: Jinhong

last: Kim

first: Zhaolin

last: Xu


title: Anti-inflammatory drugs remodel the tumor immune environment to enhance immune checkpoint blockade efficacy [SZ14]

city: Manchester

first: Agrin

last: Moeini

address: Alderley Park

country: United Kingdom

institute: Cancer Research UK Manchester Institute

summary: Identifying strategies to improve the efficacy of immune checkpoint blockade (ICB) remains a major clinical need. Here, we show that therapeutically targeting the COX-2/PGE2/EP2-4 pathway with widely used non-steroidal and steroidal anti-inflammatory drugs synergized with ICB in mouse cancer models. We exploited a bilateral surgery model to harness the heterogeneity in treatment outcome and distinguish responders from non-responders shortly following treatment. Deep cellular and molecular tumor profiling revealed acute IFN-γ-driven tumor remodeling in responder mice that was also associated with patient benefit to ICB. Crucially, monotherapy with COX-2 inhibitors or EP2-4 PGE2 receptor antagonists rapidly induced this response program and, in combination with ICB, increased the intratumoral accumulation of T cells with enhanced effector function. Inhibition of the COX-2/PGE2 pathway in patient-derived tumor fragments from multiple patients and cancer types revealed a similar shift in the tumor inflammatory environment to favor T cell activation. Our findings establish the COX-2/PGE2/EP2-4 axis as an independent immune checkpoint and a readily translatable strategy to switch the tumor inflammatory profile from cold to hot and enhance the efficacy of immunotherapy.

overall_design: Bulk RNA profiling of Braf-mutated melanoma tumours treated with vehicle, prostaglandin E2 receptor antagonists (EPAT), anti-PD-1 or anti-PD-1 plus EPAT for 2 and 5 days.

first: Victoria

middle: S

last: Pelly

first: Santiago

last: Zelenay


title: Expression data from control and infected susceptible and resistant wheat plants

status: Public on Sep 12 2021

city: Mohali

first: Jitendra

last: Kumar

state: Punjab

address: National Agri-Food Biotechnology Institute (Department of Biotechnology, Government of India) C-127, Industrial Area S.A.S. Nagar, Phase 8 Mohali-160071, Punjab, India

country: India

summary:
Comparative analysis of transcriptome in two wheat genotypes with contrasting levels of virus resistance.
We used microarrays to investigate the global gene expression in response to virus infection.

overall_design: Wheat leaf tissues were collected for RNA extraction and hybridization on Affymetrix microarrays.

first: Sudhir

last: Singh


title: Multidimensional PTEN missense variant analysis reveals variant subgroups including potential dominant negatives

city: Cleveland

first: Kenneth

last: Matreyek

state: OH

address: 2103 Cornell Rd

department: Pathology

institute: Case Western Reserve University

summary: Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. We previously developed Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. Here, we reapplied VAMP-seq to quantify the abundances of additional PTEN missense variants which were missing from the original experiment.

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: Barcoded missense variant libraries of EGFP-fused PTEN were recombined into HEK293T cells previously engineered to contain a Bxb1 recombination site at the AAVS1 locus. Upon sorting cells for EGFP expression, genomic DNA was extracted. Barcodes were amplified and counted with Illumina sequencing. Barcodes were associated back to the corresponding PTEN variant by comparison with a barcode-variant map previously created by sequencing the variant library plasmids using PacBio sequencing.     Copyright 2018 University of Washington. Data and Scores are owned by the University of Washington. Permission is hereby granted to use, reproduce, and distribute the Data and Scores for noncommercial academic research purposes only, provided that (i) credit for source and copyright are included with each copy and (ii) a link to the original material is provided whenever the material is published elsewhere on the Web. For questions regarding use by non-profit entities or commercial purposes contact: Douglas Fowler, dfowler@uw.edu

first: Douglas

middle: M

last: Fowler


title: Anti-inflammatory drugs remodel the tumor immune environment to enhance immune checkpoint blockade efficacy

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Anti-inflammatory drugs remodel the tumor immune environment to enhance immune checkpoint blockade efficacy [SZ9]

overall_design: Bulk RNA profiling of CT26 colorectal tumours treated with vehicle, celecoxib (CXB), anti-PD-1 or anti-PD-1 plus celecoxib for 7 days.


title: Genome-wide transcriptome profiling Brain-Chip and Conventional Cell Culture System Using RNA-seq

status: Public on Sep 10 2021

city: Boston

first: Iosif

last: Pediaditakis

state: MA

address: 27 Drydock avenue

institute: Emulate Inc.

summary: The goal of this study was A) to compare the global RNA-sequencing (RNA-seq) profiles data of the neurovascular units constructed in i) conventional cell cultures (n=4), ii) Substantia Nigra Brain-Chips cultured under constant flow (n=4), and iii) human adult brain-derived substantia nigra (n=8) retrieved from the Genotype-Tissue Expression (GTEx) Portal, B) to identify differences in the transcriptome profiles in brain endothelial cells  between the two conditions, αSyn fibrils and αSyn monomers.

overall_design: Global RNA expression profiles of Brain-Chips day 8 (n=4 per channel); Conventional cell culture systems day8 (n=4 upper compartment); treated Brain-Chips (n=4 vascular channel per two conditions) were generated by deep sequencing using Illumina HiSeq.

first: Dimitris

middle: V

last: Manatakis


title: A Phase II Trial of Adjuvant Durvalumab following Trimodality Therapy for Locally Advanced Esophageal and Gastroesophageal Junction Adenocarcinoma: A Big Ten Cancer Research Consortium Study

city: Indianapolis

first: Milan

last: Radovich

state: IN

address: 980 W. Walnut St. Room C240

institute: Indiana University

summary: Correlative analysis using RNA-seq was performed on tumor samples from patients with locally advanced esophageal and gastroesophageal junction adenocarcinoma treated with adjuvant durvalumab as part of a Phase II trial. The primary goal of this correlative analysis was to perform immune deconvolution using CIBERSORTx in order to determine the relative proportion of immune cell types in each sample. The immune components were then associated with relapse free survival.

overall_design: RNA-seq analysis of tumor samples from patients with locally advanced esophageal and gastroesophageal junction adenocarcinoma treated with adjuvant durvalumab


title: The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions

first: Fred

last: Winston

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5’ ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

overall_design: ChIP-Seq of Abd1-HA, Spt5-V5, and Rpb1 in wild-type and spt6-1004 cells grown at 30oC and grown in triplicate

first: Rajaraman

last: Gopalakrishnan


title: Combinatorial transcription factor profiles predict mature and functional human islet α and β cells

status: Public on Sep 09 2021

city: Nashville

first: Marcela

last: Brissova

state: TN

address: 1211 Medical Center Dr

institute: Vanderbilt University Medical Center

summary: Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled >40,000 cells from normal human islets by scRNA-seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells co-expressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-seq, MAFA/MAFB co-expressing β cells showed enhanced electrophysiological activity. Thus, these results indicate combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.

overall_design: Single cell RNAseq data of human pancreatic islets from 5 non-diabetic donors. 2 out of the 5 donors also include single cell RNAseq data from FAC-sorted alpha and beta cells. 1 out of the total 5 donors also include bulk rnaseq data.

first: Alvin

middle: C

last: Powers


title: Gene expression in murine chronic lymphocytic leukemia

status: Public on Sep 07 2021

city: Luxembourg

first: Etienne

last: Moussay

address: 6 rue Nicolas-Ernest Barblé

department: Cancer Research

country: Luxembourg

institute: Luxembourg Institute of Health

summary: mRNA profiles of B cells from 8-week-old wild-type (WT) and leukemic (Eu-TCL1) mice were generated by 3'-sequencing, in triplicate.

overall_design: mRNA profiles from B cells from WT (C57BL/6) and leukemic (Eu-TCL1) mice

first: Jerome

last: Paggetti


title: Histone H3K36me2-specific methyltransferase ASH1L promotes the MLL-AF9-induced leukemogenesis

city: East Lansing

first: Jin

last: He

state: Michigan

address: 603 Wilson Road, Biochem Bldg. Rm. 410

institute: Michigan State Univeristy

summary: ASH1L and  MLL1 are two histone methyltransferases that facilitate transcriptional activation during normal development. However, the roles of ASH1L and its enzymatic activity in the development of MLL-rearranged leukemias are not fully elucidated in Ash1L gene knockout animal models. In this study, we used an Ash1L conditional knockout mouse model to show that loss of ASH1L in hematopoietic progenitor cells impaired the initiation of MLL-AF9-induced leukemic transformation in vitro. Furthermore, genetic deletion of ASH1L in the MLL-AF9-transformed cells impaired the maintenance of leukemic cells in vitro and largely blocked the leukemia progression in vivo. Importantly, the loss of ASH1L function in the Ash1L-deleted cells could be rescued by wild-type but not the catalytic-dead mutant ASH1L, suggesting the enzymatic activity of ASH1L was required for its function in promoting MLL-AF9-induced leukemic transformation. At the molecular level, ASH1L enhanced the MLL-AF9 target gene expression by directly binding to the gene promoters and modifying the local histone H3K36me2 levels. Thus, our study revealed the critical functions of ASH1L in promoting the MLL-AF9-induced leukemogenesis, which provides a molecular basis for targeting  ASH1L and its enzymatic activity to treat MLL-arranged leukemias.

overall_design: Examine the differential gene expression in normal hematopietic progenitor cells, MLL-AF9-transformed wild-type cells, and Ash1L-knockout MLL-AF9-transformed cells

first: Mohammad

last: Aljazi


title: Analysis of developmental imprinting dynamics in primates using SNP-free methods to identify imprinting defects in cloned placenta

first: Wenhao

last: Zhang

address: Warren Alpert Bldg, Rm 160, 200 Longwood Ave

type: Methylation profiling by high throughput sequencing

summary: Our knowledge of genomic imprinting in primates is lagging behand that of mice largely due to the difficulties of allelic analyses in outbred animals. To understand imprinting dynamics in primates, we profiled transcriptomes, DNA methylomes and H3K27me3 in uniparental monkey embryos. We further developed single-nucleotide polymorphisms (SNP)-free methods, TARSII and CARSII, to identify germline differentially methylated regions (DMRs) in somatic tissues. Our comprehensive analyses showed that allelic DNA methylation, but not H3K27me3, is a major mark that correlates with paternal-biasedly expressed genes (PEGs) in uniparental monkey embryos. Interestingly, primate germline DMRs are different from PEG-associated DMRs in early embryos and are enriched in placenta. Strikingly, most placenta-specific germline DMRs are lost in placenta of cloned monkey. Collectively, our study establishes SNP-free germline DMR identification methods, defines developmental imprinting dynamics in primates and demonstrates imprinting defects in cloned monkey placenta, which provides important clues for improving primate cloning.

sample_organism:
Macaca fascicularis
Macaca mulatta

platform_organism:
Macaca fascicularis
Macaca mulatta

overall_design: We conducted RNA-seq, H3K27me3 CUT&RUN and WGBS in monkey uniparental 16-cell embryos. Also we have profiled WGBS in monkey adult tissues, WGBS and RNA-seq in monkey wild-type and SCNT placenta.

first: Chu

last: Chu

first: Yu

last: Kang

first: Weizhi

last: Ji

first: Yuyu

last: Niu

first: Yi


title: Protein kinase C (PKC)-independent signaling pathways regulate P-TEFb biogenesis in CD4+ T cells to reverse HIV latency

first: Konstantin

last: Leskov

address: 2109 Adelbert Rd., WRT205

department: Molecular Biology and Microbiology

summary: The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in latently infected memory CD4+ T cells and cellular mechanisms controlling its availability for proviral transcription are not well-defined. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we sought to identify specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Unexpectedly, cellular treatment with diacylglycerol-mimicking protein kinase C (PKC) agonists in the absence of intracellular calcium mobilization with an ionophore was sufficient to stimulate active P-TEFb expression and reactivate latent HIV with minimal cytotoxicity. Furthermore, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell RNA-seq analysis revealed that of the four known isoforms of RasGRP, the diacylglycerol-dependent Ras guanine nucleotide exchange factor RasGRP1 is by far the predominantly expressed isoform in memory CD4+ T cells and should therefore mediate the activation of ERK1/2 upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-AKT-mTORC1/2 pathway alongside the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and strongly suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells in response to either TCR co-stimulation or PKC agonist challenge is largely independent of PKC activity but rather primarily involves two complementary signaling arms of the TCR cascade namely RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-AKT-mTORC1/2.

overall_design: Approximately 1.0 X 10^7 memory CD4+ T cells were routinely isolated by negative bead selection from previously frozen healthy donor leukapheresis packs containing 1.0 X 10^8 peripheral blood mononuclear cells (PBMCs) using the EasySepTM CD4+ T-cell enrichment kit (StemCell). After isolation cells were allowed to recover overnight in complete RPMI media lacking IL-2. Purity and resting status of the isolated memory T cells were confirmed by immunofluorescence flow cytometry analysis of CD45-RO, cyclin D3 and Ki67 expression. Cells were challenged with Dynabeads CD3/CD28 to activate the T-cell receptor (TCR) at a 1:1 bead-to-cell ratio. 0.6 X 10^6 cells were taken at 24 h and 72 h after TCR stimulation for Drop-seq along with unstimulated control cells.


title: Molecular gatekeepers balancing differentiation and maintenance of human male germline stem cells uncovered by single cell resolution analysis

status: Public on Sep 08 2021

city: Münster

first: Nina

last: Neuhaus

address: Albert-Schweitzer-Campus 1

department: Centre of Reproductive Medicine and Andrology

country: Germany

institute: University Hospital of Münster

summary: Human male germline homeostasis depends on a balance between differentiation and maintenance of spermatogonial stem cells. To unveil the molecular regulation of this balance we compared human testicular tissues with severely perturbed germ cell differentiation with the normal situation. By performing single cell RNA sequencing of close to 30.000 cells and histomorphometric analysis we demonstrate that the proportion of spermatogonia remains remarkably stable despite the dwindling numbers of differentiated germ cells. In spite of this, these samples displayed altered stem cell dynamics with a push of reserve stem cells towards differentiation. This shift in dynamics was associated with the activation of gene networks regulated by four main gatekeeping transcription factors: ASCL2, EGR4, HOXC9, and DLX5. We provide invaluable information on the pathways regulating human germline stem cell dynamics along with evidence for a striking depletion of the reserve stem cell pool when these are disturbed.

overall_design: single-cell RNA-Seq of testicular tissue of patients with normal spermatogenesis (Normal; n=3) and patients with cryptozoospermia (Crypto; n=3)

first: Sara

last: Di Persio

first: Tobias

last: Tekath

first: Sandra

last: Laurentino


title: RNAseq of DHX15-silenced endothelial cells

status: Public on Sep 04 2021

city: Barcelona

first: Manuel

last: Morales-Ruiz

address: 170 Villarroel

institute: Hospital Clinic/IDIBAPS/Ciberehd

summary: We performed RNAseq in immortalized mouse-liver sinusoidal endothelial cells with or without DHX15 gene silencing. The RNA-seq experiment allowed us to generate 122,968 paired-end reads that were successfully mapped to the mouse reference GRCm38 genome assembly. After quality control analysis, four samples per experimental condition were included in the analysis.

overall_design: The mRNA profiles of two experimental conditions were compared: 1) wild-type immortalized liver sinusoidal endothelial cells and 2) immortalized liver sinusoidal endothelial cells silenced for the mRNA helicase DHX15 (n=4 for each experimental condition).

first: Jordi

last: Ribera

first: Irene

last: Portolés


title: Transcriptome profile of the zebrafish sinoatrial ring

city: Warszawa

first: Cecilia

middle: Lanny

last: Winata

address: Ks. Trojdena 4

country: Poland

institute: International Institute of Molecular and Cell Biology in Warsaw

summary: Sino Atrial Node (SAN) is part of the cardiac conduction system, which controls the rhythmic contraction of the vertebrate heart. SAN consists of a specialized pacemaker cell population that has the potential to generate electrical impulses. Although SAN pacemakers have been extensively studied in mammalian and teleost models, including the zebrafish, their molecular nature remains inadequately comprehended. Here, we utilized the zebrafish transgenic line sqet33mi59BEt to isolate cells of the sino-atrial ring (SAR) of developing zebrafish embryos and profiled their transcriptome. Our analyses identified novel candidate genes and well-known conserved signaling pathways (Wnt, BMP) involved in pacemaker development. We show that the zebrafish SAR expresses several mammalian SAN pacemaker signature genes, which include hcn4 and calcium and potassium gated channels. Moreover, genes encoding components of the BMP and Wnt signaling pathways, as well as members of the Tbx family, which have previously been implicated in pacemaker development, were also overexpressed in the SAR. Among SAR-overexpressed genes, 24 had human homologues implicated in 104 different ClinVar phenotype entries related to various forms of congenital heart diseases. Moreover, we show that loss-of-function of three SAR-overexpressed genes, pard6a, prom2, and atp1a1a.2, resulted in abnormal heart morphology and physiology. Our results suggest the relevance of our transcriptomics resource to studying human heart conditions.

overall_design: Intact hearts from transgenic line sqet33mi59BEt embryos were chemically dissociated into a single-cell suspension for FACS. Cells were sorted into GFP positive (GFP+) fraction representing sinoatrial ring and GFP negative (GFP-) fraction representing rest of heart, three biological replicates each. To identify genes enriched in sinoatrial ring, differential expression analysis were performed using the GFP- sample as background.

first: Rashid

last: Minhas


title: Analysis of differential genes in myocardial hypertrophy mice treated with dapagliflozin

status: Public on Sep 01 2021

city: Tianjin

first: Hao

last: Wu

address: No.190 Jiaoyuan Road, Hongqiao District

department: Department of Cardiology

country: China

institute: Tianjin Union Medical Center

summary:
The purpose of this study was to investigate the effect of dapagliflozin (DAPA), a sodium-glucose cotransporter 2 inhibitor, on relieving cardiac hypertrophy and its potential molecular mechanism.
Dapagliflozin activated the Plin5/PPARα signaling axis and exerts a protective effect against cardiac hypertrophy.

overall_design: the tissues from HF, HFS and NC group were sujected to total RNA isolation using TRIzol reagent. Then, a microarray analysis was taken to analyze the differential genes.

first: Jing

last: Yu


title: Comparison of transcriptomic responses to pancreas disease (PD) and heart and skeletal muscle inflammation (HSMI) in heart of Atlantic salmon

status: Public on Sep 03 2021

city: Aas

first: Aleksei

last: Krasnov

address: Osloveien 1

department: Fish Health

country: Norway

institute: Nofima AS

summary: PD and HSMI are viral diseases that cause heavy damages in Atlantic salmon aquaculture. This study was performed to examine and compare the time-courses of transcriptome responses to the causative agents - salmon alphavirus (SAV) and piscine reovirus (PRV).

overall_design: Two challenge trials were carried out and samples were collected at four (SAV) and five (PRV) time-points. Analyses were run in heart, the most infected and damaged tissue.

first: Lill

last: Johansen

first: Jorunn

last: Jørgensen


title: Heme-Oxygenase 1 is a Master Regulator of Cell Fate Following Oxidative Stress Response in Endothelial Cells

status: Public on Sep 05 2021

city: La Jolla

first: Shankar

last: Subramaniam

state: California

address: 9500 Gilman Drive

department: Bioengineering

institute: University of California, San Diego

summary:
Purpose: Excess oxidative stress (OS) impairs endothelial function and plays an important role in vascular diseases, diabetes, and neuronal disorders. Several consequences of OS including cell recovery and apoptosis have been described previously. In this study, we report systems model of the temporal dynamics of the oxidative stress response in Human Umbilical Vein Endothelial Cells (HUVECs) and characterize HMOX1 as a master regulator in orchestrating the response to oxidative stress.
Methods: Following stress induction by hydrogen peroxide (HP), we carried out dose-dependent phenotypic assays and time series measurements of transcripts in HUVECs. To explore the role of HMOX1, HUVECs were transfected with scrambled siRNA or si-HMOX1 and then treated with hydrogen peroxide. Total RNA was collected from Scrambled-untreated, scrambled-H2O2, and si-HMOX1-H2O2 samples for RNAseq. Novel findings were validated by qPCR and functional assays.
Results: In this study we have identified three phases of stress response (acute response from 1-2 hours, the transition from 4-6 hours, and the chronic stress response from 8-16 hours). We observe stable cell cycle arrest and impaired DNA replication/repair from 8-16 hours along with and increased resistance to apoptosis from 2-16 hours. Knockdown of HMOX1 prior to HP treatment releases the resistance to apoptosis. Similarly, many cellular processes switch their direction of regulation in HMOX1 deficient cells upon HP treatment (i.e. autophagy, mitochondria, cell surface signaling, expression of histone modifying enzymes and long-noncoding RNAs etc).
Conclusions: Endothelial cell fate decisions are critically important for vascular health. In this study we provide a temporal model characterizing the transition from the acute stress response to the chronic response. The nexus of cell function and dysfunction is characterized by the concurrent activation of survival and death pathways. We explore the master regulatory role of HMOX1 and identify novel mechanisms by which HMOX1 may regulate cell fate decisions.

overall_design:
After treatment with HP, samples were collected at 0 (control), 1, 2, 4, 6, 8, 10, 12, 14, and 16 hour time points in duplicate. Two biological replicates are included per time point.

After transfection with either scrambled or si-HMOX1 siRNA for 48 hours, cells were treated with HP for 6 hours before they were collected for RNAseq. Two biological replicates are included per treatment group.

first: Sindhushree

last: Raghunandan

first: Srinivasan

last: Ramachandran

first: Eugene

last: Ke

first: Yifei

last: Miao

first: Ratnesh

last: Lal

first: Zhen

last: Chen


title: C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages

city: Pittsburgh

first: Clayton

middle: Pio

last: Santiago

state: Pennsylvania

address: 1622 Locust Street

department: Ophthalmology

institute: University of Pittsburgh

summary:
Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme β-glucocerebrosidase (GCase). In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives ystemic macrophage-mediated inflammation remains incompletely understood.
To help understand the mechanisms involved, we performed gene array analysis on rC5a-treated human control and GD-induced pluripotent stem cell (iPSC)-derived macrophages.

overall_design: We analyzed gene expression changes from macrophages differentiated from iPSC lines derived from a patient with type 2 Gaucher disease or healthy controls treated with rC5a using the Affymetrix Clariom S human assay.

first: Jacquelyn

last: Serfecz

first: Afsoon

last: Saadin

first: Yuji

last: Zhang

first: Søren

last: Bentzen

first: Stefanie

middle: N

last: Vogel

first: Ricardo

last: Feldman


title: The spliceosome factor sart3 regulates hematopoietic stem/progenitor cell development in zebrafish through the p53 pathway

city: Guangzhou

first: Mei

last: Wu

state: Guangdong

address: Waihuan Dong Road

department: School of Medicine

institute: South China University of Technology

summary: Hematopoietic stem cells (HSCs) possess the potential for self-renew and the capacity, throughout life, to differentiate into all blood cell lineages. Yet, the mechanistic basis for HSC development remains largely unknown. In this study, we characterized a zebrafish smu471 mutant with hematopoietic stem/progenitor cell (HSPC) defects and found that sart3 was the causative gene. RNA expression profiling of the sart3smu471 mutant revealed spliceosome and p53 signaling pathway to be the most significantly enriched pathways in the sart3smu471 mutant. Knock down of p53 rescued HSPC development in the sart3smu471 mutant. Interestingly, the p53 inhibitor, mdm4, had undergone an alternative splicing event in the mutant. Restoration of mdm4 partially rescued HSPC deficiency. Thus, our data suggest that HSPC proliferation and maintenance require sart3 to ensure the correct splicing and expression of mdm4, so that the p53 pathway is properly inhibited to prevent definitive hematopoiesis failure. This study expands our knowledge of the regulatory mechanisms that impact HSPC development and sheds light on the mechanistic basis and potential therapeutic use of sart3 in spliceosome-mdm4-p53 related disorders.

overall_design: The sart3 mutant and the WT sibling were generated from sart3+/- intercross embryos by high-resolution melting genotyping. Then, RNA from sart3 mutant and the WT sibling  embryos was extracted with TRIzol reagent.

first: Yan

last: Zhao

first: Yiyue


title: FAK activity alters the transcriptional landscape during SMC phenotypic switching

status: Public on Sep 02 2021

city: Mobile

first: Steve

last: Lim

state: AL

address: 5851 USA Drive North MSB 2320

department: Biochemistry and molecular biology

institute: University of South Alabama

summary: Purpose: The goals of this study are to compare the effect of FAK activity on gene expression of NGS-derived RNA-sequencing during vascular remodeling.

overall_design: Methods: Mice were treated with or without FAK-I for 2 weeks after wire injury. Femoral arteries were isolated, and the fat and adventitia layers were carefully removed under surgical microscope. To remove endothelial and immune cells, the artery lumen was quickly flushed with 150 µl TRIzol lysis reagent (Invitrogen) using a 29-gauge insulin syringe. Media were frozen in liquid nitrogen and lysed in 500 µl TRIzol  per artery, and total RNA was isolated using the RNeasy column. Isolated RNA submitted for NGS library generation and sequencing. All experiments performed in triplicate.

first: Ssang-Taek

first: Kyuho

last: Jeong

first: James

last: Murphy

first: Eun-Young

middle: E

last: Ahn


title: RNA-Seq provides new insights into the nitrogen response of Azoarcus olearius BH72 during N2-fixation beyond the nitrogen fixation process

status: Public on Sep 06 2021

city: Bremen

first: Shanmugam

last: Solaiyappan Mani

address: Leobner strasse

department: Department of Microbe-Plant Interactions

institute: University of Bremen

summary: Azoarcus olearius BH72 is a diazotrophic endophyte carrying out biological nitrogen fixation (BNF) and supplying nitrogen to its host plant. Our previous microarray approach provided insights in the transcriptome of strain BH72 under N2-fixation in comparison to ammonium-grown conditions, which already indicated induction of genes not related to the BNF process. Due to the known limitations of the technique, we might have missed additional differentially regulated genes (DEGs). Thus we used directional RNA-Seq to better comprehend the transcriptional landscape under these growth conditions. RNA-Seq detected almost 24 % of the annotated genes to be regulated, twice the amount identified by microarray. In addition to confirming entire regulated operons containing known DEGs, the new approach detected induction of genes involved in carbon metabolism and flagellar and twitching motility. On the other hand, genes encoding for proteins involved in translation and vitamin biosynthesis were detected to be suppressed. Nonetheless, strain BH72 appears not to be content with N2-fixation but is primed for alternative economic N-sources, such as nitrate, urea or amino acids: we detected strong induction of machineries for uptake and assimilation.

overall_design: Directional RNA-Seq analysis to compare the transcriptome of diazotrophic Azoarcus olearius BH72 grown microaerobically under N2-fixing and non-fixing conditions (with ammonium chloride as nitrogen source). Cells and RNA were obtained from three independent experiments for each condition.

first: Barbara

last: Reinhold-Hurek


title: Gene expression in murine chronic lymphocytic leukemia B cells

first: Etienne

last: Moussay

summary: mRNA profiles were generated by 3'-sequencing, in triplicate from B cells of control mice (AHRWT/WT Hif1aWT/WT), AHR conditional KO (AHRfl/fl), Hif1a conditional KO (Hif1afl/fl) or double AHR and Hif1a conditional KO (AHRfl/fl Hif1afl/fl) .

overall_design: mRNA profiles of sorted leukemic B cells from Emu-TCL1 CD19Cre mice and from 3 different CD19 conditional KO for AHR, Hif1a, or both)

first: Jerome

last: Paggetti


title: Differentially expressed miRNAs for sebaceous gland carcinoma of the eyelid and TP53 mutation

first: chuanli

last: zhang

address: Fu kang Road251,Nankai Strict

institute: Tianjin Medical University Eye Hospital

summary: To screen the related miRNAs in the occurence and development of sebaceous gland carcinoma(SGC) of the eyelid , we have employed Human Agilient microarray expression profiling as a discovery platform to identify differentially expressed miRNAs for SGC and TP53 mtation related miRNAs. Human formalin-fixed paraffin-embedded (FFPE) SGC tissues and para-carcinoma sebaceous gland(SG) FFPE samples  from SGC patients were screened using microarray assay.  Human FFPE SGC tissues containing TP53 mutation and human FFPE SGC tissues without TP53 mutation  were screened using microarray. The overlap of two sets of microarray differentially upregulated miRNAs were selected as our research object.

overall_design: SGC of eyelid tissues with TP53 mutation contained four repeat samples and SGC of eyelid tissues without TP53 mutation also contained four repeat samples for each experiment.Differentially expressed miRNAs between human SGC of eyelid tissus and para-carcinoma tissues were screened ,and eight repeat samples were performed  for each experiment.

first: Chuanli

first: Limin

last: Zhu

first: Xun

last: Liu


title: Transcriptional profiling of equine endometrium before, during and after capsule disintegration during normal pregnancy and after oxytocin-induced luteostasis in non-pregnant mares

city: Neustadt

first: Claudia

last: Klein

address: Höltystr. 10

institute: Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics

summary: mRNA profiles of (1) equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares and (2) equine endometrium collected 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin.

overall_design: Examination of transcriptional profiles transcriptional profiles of equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares. In addition, the transcriptomes of endometrial samples obtained 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin, were determined and compared

first: Phoebe

last: Bruce

first: Jutta

last: Hammermueller

first: Tony

last: Hayes

first: Brandon

last: Lillie

first: Keith

last: Betteridge


title: Systematic research specimen collection during clinical image-guided breast biopsy does not increase complications and enables high-content analyses for translational cancer research

first: Simona

last: Cristea

state: Massachusetts

address: 450 Brookline Ave.

department: Biostatistics and Computational Biology

institute: Dana-Farber Cancer Institute

summary: Systematic collection of fresh tissues for research at the time of diagnostic image-guided breast biopsy has the potential to fuel a wide variety of innovative studies. Here we report the initial experience, including safety, feasibility and laboratory proof-of-principle, with the collection and analysis of research specimens obtained via breast core needle biopsy immediately following routine clinical biopsy at a single institution over a 14-month period. Study eligibility criteria consisted of patients age 18 or older who were undergoing a clinical breast needle biopsy. Patients providing informed consent underwent one or two additional core biopsies following collection of all necessary clinical specimens. In total, 395 patients were approached and 270 consented to the research study, yielding a 68.4% consent rate. Among consenting patients, 238 lesions were biopsied for research, resulting in 446 research specimens collected. No immediate complications were observed.  During the same period, zero complications were reported among the non-research procedures (1622 breast biopsy procedures). Representative research core specimens showed high diagnostic concordance with clinical core biopsies. Flow cytometry demonstrated consistent recovery of hundreds to thousands of viable cells per research core. Among a group of HER2+ tumor research specimens, HER2 assessment by flow cytometry correlated highly with immunohistochemistry (IHC) staining, and in addition revealed extensive inter- and intra-tumoral variation in HER2 levels of potential clinical relevance. Suitability for single-cell transcriptomic analysis was demonstrated for a triple-negative tumor core biopsy, revealing substantial cellular diversity in the tumor immune microenvironment, including a prognostically relevant T cell subpopulation. Thus, collection of fresh tissues for research purposes at the time of diagnostic breast biopsy is safe, feasible and efficient, and may provide a high-yield mechanism to generate a rich tissue repository for a wide variety of cross-disciplinary research.

overall_design: single cell RNA-seq was performed for 1 sample

first: Siang-Boon

last: Koh

first: Franziska

last: Michor

first: Leif

middle: W

last: Ellisen


title: Vertical sleeve gastrectomy triggers fast β-cell recovery upon overt diabetes

city: Neuherberg

first: Heiko

last: Lickert

address: Ingolstaedter Landstraße 1

department: Institute of Diabetes and Regeneration Research

institute: Helmholtz Zentrum München

summary: This dataset consists of single-cell RNA-seq (10X) data of pancreatic islets isolated from healthy and diabetic db/db mice that underwent VSG surgery, Sham surgery (Control) and Sham surgery + pair-feeding (Calorie-restriction control).

overall_design: 7 single-cell RNA sequencing experiments of pancreatic islets isolated from male Lepr-deficient db/db and WT mice, 16-18 weeks of age, were performed using 10X Genomics technology to generate single-cell transcriptomic profiles of healthy and db/db diabetic mice undergoing surgical interventions.

first: Lena

last: Oppenländer

first: Subarna

last: Palit

first: Kerstin

last: Stemmer

first: Michael

last: Sterr

first: Annette

last: Feuchtinger

first: Tobias

last: Greisle

first: Anika

last: Böttcher

last: Ansarullah

first: Fabian

last: Theis


title: Single cell RNA-seq analysis links respiratory dysfunction to an extracellular matrix-specific damage response in cartilage

city: Cologne

first: Bent

last: Brachvogel

address: Joseph-Stelzmann Street 52

institute: University of Cologne

summary: Energy metabolism and extracellular matrix function are closely connected to orchestrate and maintain tissue organization, but the crosstalk is poorly understood. Here, we used scRNA-seq analysis to uncover the importance of respiration for extracellular matrix homeostasis in mature cartilage. A combined approach of high-resolution single cell RNA sequencing, mass spectrometry/matrisome analysis and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. Genetic inhibition of respiration in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocyte clusters. These changes were associated with alterations in extracellular matrix composition, a shift in the collagen/non-collagen protein content and an increase of collagen crosslinking and ECM stiffness. The results demonstrate, based on findings of the scRNA-seq analysis, that respiration is a key factor contributing to ECM integrity and mechanostability in cartilage and presumably also in many other tissues.

overall_design: Examination of the single cell transcriptome in cartilage with respiration dysfunction


title: Cohesin core complex gene dosage contributes to germinal center derived lymphoma phenotypes and outcomes [single-cell RNA-seq]

city: New York City

first: Christopher

middle: Russell

last: Chin

state: NY

address: 413 E 69th Street, Belfer Building, BB-1462

institute: Weill Cornell

summary: The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 deficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 deficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

overall_design: Single cell RNA sequencing in WT and Smc3 Het mice

first: Martin

last: Rivas

first: Ceyda

last: Durmaz

first: Ari

last: Melnick


title: Discovery of synthetic lethal and tumor suppressive paralog pairs in the human genome

status: Public on Aug 31 2021

city: Seattle

first: Alice

last: Berger

state: WA

address: 1100 Fairview Ave N

institute: Fred Hutchinson Cancer Center

summary: CRISPR knockout screens have accelerated the discovery of important cancer genetic dependencies. However, traditional CRISPR-Cas9 screens are limited in their ability to assay the function of redundant or duplicated genes. Paralogs in multi-gene families constitute two-thirds of the protein-coding genome, so this blind spot is the rule, not the exception. To overcome the limitations of single gene CRISPR knockout screens, we developed paired guide RNAs for Paralog gENetic interaction mapping (pgPEN), a pooled CRISPR/Cas9 approach which targets over a thousand duplicated human paralogs in single knockout and double knockout configurations. We applied pgPEN to two cell lineages and discovered that over 10% of human paralogs exhibit synthetic lethality in at least one cellular context. We recovered known synthetic lethal paralogs such as MAP2K1/MAP2K2, important drug targets such as CDK4/CDK6, and numerous other synthetic lethal pairs such as CCNL1/CCNL2. In addition, we identified ten tumor suppressive paralog pairs whose compound loss promotes cell growth. These findings identify a large number of previously unidentified essential gene families and nominate new druggable targets for oncology drug discovery.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Paired guide RNA CRISPR screen to identify genetic interactions among human duplicate gene pairs (paralogs)

first: Phoebe

last: Parrish

first: James

middle: D

last: Thomas

first: Austin

last: Gabel

first: Shriya

last: Kamlapurkar

first: Robert

middle: K

last: Bradley


title: Iron loading induces cholesterol synthesis and sensitizes endothelial cells to TNFα-mediated apoptosis

city: Los Angeles

first: Allison

last: Fisher

state: CA

address: 650 Charles E Young Dr South, CHS 44-100

department: Medicine

institute: UCLA Center for Iron Disorders

summary: In plasma, iron is normally bound to transferrin, but in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non-transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate, FAC). Using an unbiased approach, we showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing SREBP2-mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by TNFα. Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFa-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.

overall_design: Primary human umbilical vein endothelial cells (ATCC #PCS-100-013) were treated with solvent or 100 uM ferric ammonium citrate for 30 hours. For each group we have 3 replicates from 3 independent experiments. The total number of samples is 6. Total RNA was isolated and subjected to RNA-Seq.

first: Daniel

last: Srole

first: Nicolaos

middle: J

last: Palaskas

first: David

last: Meriwether

first: Srinivasa

middle: T

last: Reddy

first: Tomas

last: Ganz

first: Elizabeta

last: Nemeth


title: Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation

city: Memphis

first: Tian

last: Mi

address: 262 Danny Thomas Pl MS351

department: Immunology

institute: St. Jude Children's Research Hospital

summary: CD8 T cell memory differentiation endows T cells with an ability to rapidly induce effector functions upon pathogen re-encounter. While it is well established that substantial epigenetic remodeling occurs during the effector stage of the immune response, the signaling events that imprint CD8 T cells with these stable epigenetic programs are not well-defined. To gain insight into the signaling determinants of effector-associated epigenetic programming among CD8 T cells, we explored the role of IL-12 in the imprinting of IFNg expression during human CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naïve CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promotor. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, resulted in significant and stable demethylation of the IFNg locus that was commensurate with an increase in IFNg expression. We further show that IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human CAR T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector epigenetic programming in CD8 T cells during the primary immune response and serve as proof of concept that signal 3 cytokines can be used to guide the induction of epigenetically regulated traits among T cells used for adoptive immunotherapies.

overall_design: 2 samples of control naïve T cells, 3 samples of naïve T cells treated with TCR, 3 samples of naïve T cells treated with TCR and IL12, 4 samples of TET2 KO and mCherry control samples at Week1 or Week2

first: Caitlin

last: Zebley

first: Maureen

last: McGargill

first: Giedre

last: Krenciute

first: Ben

last: Youngblood


title: Chemical inhibition of APE1 redox and DNA repair functions affects the inflammatory response via different but overlapping mechanisms.

status: Public on Aug 29 2021

city: Natal

first: Thais

middle: Teixeira

last: Oliveira

state: Rio Grande do Norte

address: Rua das biociências

department: Departamento de Biologia Celular e Genética

country: Brazil

institute: Federal University of Rio Grande do Norte, Brazil,

summary: The presence of oxidized DNA lesions, such as 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic sites (AP sites), has been described as epigenetic signals that are involved in gene expression control. In mammals, Apurinic-apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is the main AP endonuclease of the base excision repair (BER) pathway and is involved in active demethylation processes. In addition, APE1/Ref-1, through its redox function, regulates several transcriptional factors. However, the transcriptional control targets of each APE1 function are not completely known. In this study, a transcriptomic approach was used to investigate the effects of chemical inhibition of APE1/Ref-1 redox or DNA repair functions by E3330 or methoxyamine (MX) in an inflammatory cellular model. Under lipopolysaccharide (LPS) stimulation, both E3330 and MX reduced the expression of some cytokines and chemokines. Interestingly, E3330 treatment reduced cell viability after 48 h of the treatment. Genes related to inflammatory response and mitochondrial processes were downregulated in both treatments. In the E3330 treatment, RNA processing and ribosome biogenesis genes were downregulated, while they were upregulated in the MX treatment. Furthermore, in the E3330 treatment, the cellular stress response was the main upregulated process, while the cellular macromolecule metabolic process was observed in MX-upregulated genes. Nuclear respiratory factor 1 (NRF1) was predicted to be a master regulator of the downregulated genes in both treatments, while the ETS transcription factor ELK1 (ELK1) was predicted to be a master regulator only for E3330 treatment. Decreased expression of ELK1 and its target genes and a reduced 28S/18S ratio were observed, suggesting impaired rRNA processing. In addition, both redox and repair functions can affect the expression of NRF1 and GABPA target genes. The master regulators predicted for upregulated genes were YY1 and FLI1 for the E3330 and MX treatments, respectively. In summary, the chemical inhibition of APE1/Ref-1 affects gene expression regulated mainly by transcriptional factors of the ETS family, showing partial overlap of APE1 redox and DNA repair functions, suggesting that these activities are not entirely independent. This work provides a new perspective on the interaction between APE1 redox and DNA repair activity in inflammatory response modulation and transcription.

overall_design: The cells were grouped as follows: LPS-stimulated (LPS), LPS + 100 µM  E3330 (LPS+E3330), and LPS + 6 mM MX (LPS+MX). The LPS group was used as reference sample

first: Fabrícia

middle: L

last: Fontes-Dantas

first: Rayssa

first: Daniele

last: Pinheiro

first: Leonam

middle: G

last: Coutinho

first: Vandeclecio

last: Silva

first: Sandro

last: De Souza

first: Lucymara

last: Agnez-Lima


title: Identification of two subsets of murine Kupffer cells

city: Singapore

first: Florent

last: Ginhoux

address: 8A Biomedical Grove, Immunos Building, Level 3

country: Singapore

institute: Singapore Immunology Network (SIgN), A*STAR

summary: Tissue macrophages are immune cells whose phenotypes and functions are dictated by their origins and tissue niche. However, tissues are complex environments and macrophage heterogeneity within a same organ has been largely overlooked so far. Here, we used high-dimensional single cell sequencing to characterize macrophage populations in the murine liver. We identified two distinct populations of Kupffer cells (KCs).

overall_design: Examination of inherent murine Kupffer cell population heterogeneity

first: Camille

last: Bleriot

first: Xiao

last: Zhang

first: Jin

last: Chen


title: Impact of substrate adhesiveness on embryonic stem cell pluripotency

city: Cambridge

first: Celine

last: Labouesse

address: Puddicombe Way

department: Cambridge Stem Cell Institute

institute: University of Cambridge

summary: GOAL: We developed novel hydrogel substrates for stem cell culture, in which we independently vary substrate stiffness and adhesiveness. Substrate adhesiveness is here defined by the density of matrix tethering sites on the hydrogel surface (not by protein concentration). Any extra-cellular matrix protein can be covalently bonded to the gel at those tethering sites. The density of matrix tethering sites is controlled by the concentration of a co-factor added to the polyacrylamide hydrogel premix. The goal of this study was to assess the impact of varying matrix tethering on the gene expression of pluripotent embryonic stem cells, and to determine whether the potential effect would be dependent on substrate stiffness.

overall_design: 6 different conditions were used:  2 stiffnesses "SOFT" and "STIFF"  (E~750Pa and E~160kPa respectively)  3 adhesiveness conditions corresponding to LOW (16mM), MID (48mM) and HIGH (80mM) concentrations of the AHA co-factor.

first: Céline

first: Moritz

last: Hofer

first: Bao

last: Xiu Tan

first: Kevin

last: Chalut


title: Genome-wide impact of substrate stiffness on embryonic stem cell pluripotency and the signalling pathways controlling it

summary: GOAL:  The goal of this study was to assess the impact of substrate stiffness on the gene expression of pluripotent embryonic stem cells, in different heterogeneous conditions. Embryonic stem cells cultured on tissue culture plastic have heterogeneous gene expression when cultured in serum+LIF. We investigated whether culturing cells on soft fibronectin-coated StemBond hydrogels has genome-wide effects and in particular improves the homogeneity of naive pluripotent gene expression. To assess the interplay with multiple signalling pathways, we used multiple pluripotent conditions or differentiating conditions, by adding or removing the MEK inhibitor PD03, and the cytokine LIF.

overall_design: Embryonic stem cells were cultured on fibronectin-coated StemBond hydrogels and tissue culture plastic for either 24hrs or 48hrs. Culture conditions were multiple: serum+LIF (24hrs), serum+LIF (48hrs), serum without LIF (48hrs), serum+PD03 (48hrs), serum+LIF+PD03 (48hrs). 3 replicate samples per condition were sequenced.

first: Chibeza

last: Agley

first: Giuliano

last: Stirparo

first: Paul

last: Bertone


title: RNA sequencing of patient derived xenograft sample ALL-811 using prime-seq

status: Public on Aug 28 2021

city: Munich

first: Irmela

last: Jeremias

address: Feodor-Lynenstr. 21

department: Research Unit Apoptosis in Hematopoietic Stem Cells

institute: Helmholtz Zentrum München, German Center for Environmental Health (HMGU), Munich, Germany

summary: We establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of DUX4 (double homeobox 4) playes an essential role in patients’ leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability.

overall_design:
DUX4 KD and control PDX cells were isolated out of the murine bone marrow after induction of the shRNA expression by tamoxifen. the mRNA expression pattern was analyzed.
>>> Raw data not provided as German law does not allow human raw sequence data to be publicly available. <<<

first: Jeremias

last: Irmela

first: Carlet

last: Michela

first: Kerstin

last: Völse

first: Martin

last: Becker

first: Johannes

last: Bagnoli


title: Determining the mechanosensitive targets of ERK and Wnt pathways in embryonic stem cells

summary: Embryonic stem cells can be maintained in a state of naive pluripotency by the use of 2 inhibitors, PD03 - a MEK inhibitor upstream of ERK, and CHIRON, a GSK3 b inhibitor, which acts as a Wnt agonist. The removal of these inhibitors allow embryonic stem cells to exit pluripotency. To determine if targets of transcriptional targets of these pathways are mechanosensitive such that their activation or suppression would be dependent on mechanical properties of substrate.

overall_design: Embryonic stem cells were cultured in pluripotency conditions 2i+LIF on soft or stiff StemBond hydrogels for 12 hrs. Then PD03 or CHIRON were removed for 2hrs and 12hrs before collecting RNA lysates for sequencing, and control samples remained in 2i+LIF served as 0hr timepoint . 2 replicates per condition were sequenced.

first: Christophe

last: Verstreken

first: Michael

middle: A

last: Barber


title: RNAseq analysis of human CD14+ monocytes exposed or not to TNFa

city: Paris

first: Elodie

last: Segura

address: 156 rue de Vaugirard

department: INSERM U1151

country: France

institute: INSERM

summary: Human CD14+ monocytes were cultured with M-CSF and IL-4 with or without TNF-a for 3h or 6h.

overall_design: 23 samples

first: Alba

last: De Juan

first: Alice

last: Coillard


title: Profiling circular RNA in esophageal squamous cell carcinoma (ESCC) with lymph node metastasis

city: Nanjing

first: Gaochao

last: Dong

address: Baiziting 42, Nanjing, P.R. China

department: Jiangsu Key Laboratory of Molecular and Translational Cancer Research

institute: The Affiliated Cancer Hospital of Nanjing Medical University,

summary: Circular RNAs (circRNAs) have been identified as a novel class of non-conding RNAs, which are stable and have covalently single-stranded loop configuration.With the improvement of RNA-sequencing and bioimofrmatics, numerous dysregulated expression of circRNAs has been identified in kinds of physiological and pathological processes, especially in tumorigenesis. To date, however, the discovery of cirtical circRNA abnormalities, as well as their functions and underlying mechanisms, in the progress of esophageal squamous cell carcinoma (ESCC), is still unknown.

overall_design: Microarray expression profiling of circRNAs were conducted using RNA extracted from three esophageal squamous cell carcinoma (ESCC) tissues, three matched adjacent normal samples, and three matched metastatic lymph node.

first: Feng

last: Jiang

first: Yingkuan

last: Liang


title: TGF-β Family Signaling Promotes CDK7 Inhibitor Resistance in Triple Negative Breast Cancer Cells through Upregulation of Multidrug Transporters

status: Public on Aug 27 2021

city: Cleveland

first: Ruth

last: Keri

address: 9500 Euclid Avenue

department: Cancer Biology

institute: Cleveland Clinic Lerner Research Institute

summary: Identifying a mechanism by which CDK7 inhibitor resistance occurs in TNBC

overall_design: RNA-seq: Three replicates were analyzed for each experimental condition.

first: Bryan

middle: M

last: Webb


title: Cohesin core complex gene dosage contributes to germinal center derived lymphoma phenotypes and outcomes [bulk RNA-seq]

first: Christopher

last: Chin

overall_design: Bulk RNA sequencing in WT and Smc3 Het mice

last: Rivas

first: Ceyda

last: Durmaz

middle: R

first: Ari

last: Melnick


title: TDP-43 represses a cryptic exon in Schwann cells to ensure rapid saltatory conduction

first: Shuo-Chien

last: Ling

state: Singapore

address: 12 Science Drive 2, MD1, 16-03-H

department: Physiology

institute: National University of Singapore

summary: A pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia is the aggregation of TDP-43. While extensive exploration into the function of TDP-43 has focused entirely in the central nervous system (CNS), the implications in physiology and pathology of the peripheral nervous system (PNS) have long been overlooked. Herein, we demonstrate that deletion of TDP-43 in Schwann cells results in a 50% reduction in peripheral nerve conduction velocity, without any obvious alterations to peripheral compact myelin. Compromised insulatory function of myelin was due to the complete loss of paranodal axoglial junctions flanking the nodes of Ranvier. By contrast, the paranodal junctions in the CNS oligodendrocytes are unaltered upon deletion of TDP-43. Mechanistically, TDP-43 binds directly to Nfasc mRNA, which encodes neurofascin, a key cell adhesion molecule essential for establishing paranodal junctions. TDP-43 binding sites overlap with a cryptic exon of Nfasc such that loss of TDP-43 leads to the retention of this cryptic exon with a premature stop codon, a prerequisite for nonsense-mediated decay. Thus, TDP-43 is required for the proper assembly of nodes of Ranvier that is essential for saltatory conduction by suppressing the cryptic exon in the Nfasc mRNA of the Schwann cells. Our findings indicate a direct involvement of TDP-43 in normal PNS function and suggest that PNS-autonomous dysfunction in saltatory conduction may contribute to TDP-43 proteinopathies.

overall_design: Bulk RNA-Seq of sciatic nerve tissue of 21-day old mice with TDP-43 deleted in Schwann cells.

first: Kae-Jiun

last: Chang

first: Ira

last: Agrawal

first: Wan

middle: Y

last: Ho

first: Jonah

last: Chan


title: RNA sequencing of patient derived xenograft sample AML-388 using prime-seq

first: Irmela

last: Jeremias

summary: We establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities.

overall_design:
MCL1 KD and control PDX cells were isolated out of the murine bone marrow after induction of the shRNA expression by tamoxifen. the mRNA expression pattern was analyzed.
Please note that the raw data is not provided as the German law does not allow raw sequencing data to be publicly available.

first: Jeremias

last: Irmela

first: Carlet

last: Michela

first: Kerstin

last: Völse

first: Johannes

last: Bagnoli


title: ATAC-seq and RNA-seq of hematopoietic progenitor cells (HPC) and Natural Killer (NK) cells transduced with transcription factors T-BET or EOMES

city: Ghent

first: Wouter

last: Van Loocke

state: East Flanders

address: C. Heymanslaan 10

department: Biomolecular Medicine

country: Belgium

institute: Ghent University

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Molecular predictors of response to pembrolizumab in thymic carcinoma

city: Washington

first: Krithika

last: Bhuvaneshwar

state: DC

address: 2115 Wisconsin Ave NW

institute: Georgetown University

summary: Thymic carcinoma is rare and has a poorer prognosis than thymomas. The treatment options are limited after failure of platinum-based chemotherapy. We previously performed a single center phase II study of pembrolizumab in patients with advanced thymic carcinoma, showing a 22.5% response rate. Here, we characterized the genomic and transcriptomic profile of thymic carcinoma samples from 10 patients (5 non-responders vs 5 responders) in this cohort, with the main aim of identifying potential predictors of response to immunotherapy. We found that expression of PDL1 and alterations in genes or pathways that correlated with PD-L1 expression (CYLD and BAP1) could be potential predictors for response or resistance to immunotherapy in patients with advanced thymic carcinoma. Our study provides insights into potential predictive markers/pathways to select patients with thymic carcinoma for anti-PD-1 immunotherapy.

overall_design: In this paper, we characterized the genomic profile of 10 patient samples (5 responders vs 5 non-responders) using whole exome sequencing and whole transcriptome sequencing.

first: Yongfeng

last: He

first: Archana

last: Ramesh

first: Yuriy

last: Gusev

first: Giuseppe

last: Giaccone


title: Next Generation Sequencing of kidneys 24hours after ischemia-reperfusion injury performed in conscious mice or under anesthesia

first: Pierre

last: Galichon

address: 4 rue de la Chine

department: Hopital Tenon - Urgences Nephrologiques et Transplantation Renale

institute: Assistance Publique - Hopitaux de Paris

summary: We study the effect of genereal anesthesia on the transcriptomic changes induced by ischemia-reperfusion injury in the kidney

overall_design: Kidney mRNA profile of 4 control mice, 4 mice 24 hours after ischemia-reperfusion performed in conscious mice, 4 mice 24 hours after ischemia reperfusion performedunder general anesthesia

first: David

last: Legouis

first: Juliette

last: Hadchouel

first: Charles

last: Verney


title: small RNA expression in lung extracellular vesicles (EV) from C57BL/6J mice exposed to ozone

city: Chapel Hill

first: Gregory

middle: James

last: Smith

state: North Carolina

address: 120 Mason Farm Road, 5070E Genetic Medicine Building

department: Genetics

institute: University of North Carolina

type: Non-coding RNA profiling by high throughput sequencing

summary: We performed small RNA sequencing on extracellular vesicles isolated from bronchoalveolar lavage fluid (BALF) collected from ozone and filtered air exposed C57BL/6J mice

overall_design: Test for effect of inhalation exposure to ozone on lung EV small RNA expression

first: Samir

last: Kelada


title: RNAseq of Kupffer cells treated with IL2 and purifed Kupffer cells sub-populations (Kupffer Cell 1 and Kupffer Cell 2)

city: Milan

first: Jose

middle: Manuel

last: Garcia-Manteiga

address: Via Olgettina 58

department: Center for Translational Genomics and Bioinformatics

country: Italy

institute: OSR - San Raffaele Scientific Institute

summary: Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages long known for their scavenger and phagocytic functions. KCs are also able to present antigen to CD8+ T cells and promote either T cell tolerance or full effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection – where HBV-specific naïve CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction – to identify a subset of KCs (referred to as KC2) that improves the antiviral function of T cells upon exogenous administration of IL-2. Mechanistically, KC2 were found to be both enriched in the IL-2 sensing machinery and poised to productively cross-present hepatocellular antigens in response to this cytokine. Removing MHC-I from all KCs – including KC2 – as well as selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. Together, these findings indicate that, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment and suggest new strategies for boosting T cell immunity in the liver.

overall_design: Isolated Kupffer Cells from C57BL/6 mice were treated with either PBS or IL2. In addition, isolated Kupffer Cells from C57BL/6 mice where further sorted in two sub-populations, KC1 and KC2, based on surface markers ESAM and CD206. 3 different biological replicates where obtained for each KC group. No treatment was performed on KC1/KC2

first: Giorgia

last: De Simone

first: Francesco

last: Andreata

first: Valeria

last: Fumagalli

first: Camille

last: Bleriot

first: Florent

last: Ginhoux

first: Matteo

last: Iannacone

first: Stefano

last: Gilotto

first: Chiara

last: Laura

first: José


title: Rett Syndrome linked to defects in forming the MeCP2/Rbfox/LASR complex in mouse models

city: Shanghai

first: Xing

last: Fu

address: 3888 Chenhua Road

institute: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences

summary: Rett syndrome (RTT) is a severe neurological disorder which is mainly caused by mutations found in the X-linked gene encoding MeCP2. Despite extensive studies, the molecular functions of MeCP2 remain elusive. Here, we report that MeCP2 is a new subunit of a higher-order multiunit protein complex Rbfox/LASR and acts as a scaffold for this splicing complex. Deletion or mutation of MeCP2 leads to defects in forming MeCP2/Rbfox/LASR complex and aberrant alternative pre-mRNA splicing. Our data link RTT to an impaired function of MeCP2 in splicing control through its role in nucleating Rbfox/LASR macromolecule assembly.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: In this study, we performed RNA-seq, ChIP-seq and iCLIP-seq using HEK293T cells or mouse brain tissues.

first: Yan

last: Jiang

first: Jingyi

last: Hui


title: In vivo structure and dynamics of the RNA genome of SARS-Cov-2

city: Beijing

last: Zhang

state: Not Applicable

address: Dongdajie street

institute: Beijing institute of Biotechnology

summary: The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3’-UTR and 5’-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide the first evidence of comprehensive interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we find alternative short and long distance arches around FSE, forming a “high-order pseudoknot” embedding FSE, which might help ribosome stalling at frameshift sites. We found that during packaging, SARS-CoV-2 genome RNA undergoes compaction while genome domains remain stable and genome cyclization is weakened. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting describes dynamics on RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies.

sample_organism: Severe acute respiratory syndrome coronavirus 2

overall_design: We developed a simplified SPLASH protocol to capture RNA-RNA interactions in the SARS-CoV-2 virus. Briefly, the biological samples are first stabilized by Psoralen-PEG3-Biotin cross-linking, followed by RNase III treatment, proximity ligation, library preparation, and sequencing. Samples were collected from different phases of the SARS-CoV-2 virus viral cycle to infer the dynamic structure of viral RNA at different stages. In the early stage of infection, we collected virus-infected Vero cells (C), in which cytopathic effect (CPE) was not observed. In the later stage of infection, when 70% cells underwent CPE, we collected the cell culture supernatant and harvested virus particles (V) and, at the same time used freeze-thaw methods to lyse the cells (L) to collect the cell and virus RNA

first: Kun

last: Huang

first: Dejian

last: Xie

first: Lau

last: You

first: Wenlong

last: Shen

first: Ping

last: Li

first: Dong

last: Wang

first: Zhong

last: Zou

first: Meilin

last: Jin

first: Grzegorz

last: Kudla

first: Zhihu

last: Zhao


title: StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells

first: Celine

last: Labouesse


title: Reinforcement of transcriptional silencing by a positive feedback between DNA methylation and non-coding transcription

city: Ann Arbor

first: Andrzej

middle: T.

last: Wierzbicki

state: MI

address: 1105 N. University Ave.

department: Department of Molecular, Cellular and Developmental Biology

institute: University of Michigan

summary: Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNA. This facilitates locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription, which is consistent with a mutual reinforcement of DNA methylation and non-coding transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that loss of DNA methylation leads to a strong reduction of Pol V transcription. This occurs when DNA methylation is lost in all sequence contexts, which may happen not only in mutants defective in RdDM but also in mutants lacking maintenance DNA methyltransferases. Our results support a model where RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription with a strong crosstalk with other silencing pathways.

overall_design: RNA immunoprecipitation with an anti-NRPE1 antibody was performed on 5 samples, which include 3 different genotypes (cmt3:2replicates, drm2: 2replicates, met13)

first: M

middle: H

last: Rothi

first: Masayuki

last: Tsuzuki

first: Shriya

last: Sethuraman


title: Profiling of the chromatin landscape of human natural killer (NK) cells overexpressing the transcription factors T-BET or EOMES

first: Wouter

last: Van Loocke

summary: T-BET and EOMES are key transcription factors in the development of mature NK cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES in human umbilical cord blood-derived hematopoietic progenitor cells (HPC) and cultured them in vitro in an NK cell differentiation model. On day 21 of culture mature stage 4 (CD56+CD94+CD16-) and stage 5 (CD56+CD94+CD16+) NK cells from T-BET or EOMES overexpression and control cultures were sorted, whereafter genomic DNA was isolated and the chromatin accessibility landscape was determined by assay for transposase-accessible chromatin (ATAC) sequencing. Profiling of the epigenetic changes during T-BET or EOMES overexpression in mature NK cells revealed new insights in the regulatory role of T-BET and EOMES during terminal NK cell maturation.

overall_design: Profiling of the chromatin accessibility landscape by Fast-ATAC sequencing of stage 4 and stage 5 NK cells with T-BET or EOMES overexpression in comparison to control NK cells on day 21 of culture.

first: Laura

last: Kiekens

first: Georges

last: Leclercq


title: Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas

city: La Jolla

first: Udit

last: Parekh

address: 9500 Gilman Drive, Room 353, Powell-Focht (EBU3A)

department: Bioengineering

institute: University of California, San Diego

summary: To deconstruct tissue-specific effects of genes and variants on proliferative advantage and transformation potential we developed a novel approach, harnessing single-cell cancer driver screens in teratomas coupled with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and gene variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serially injected teratomas. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. These drivers directed teratoma development to lineages representative of pediatric tumors such as medulloblastoma and rhabdomyosarcoma. Additionally, mutant MEK1 (S218D/S222D) provides a proliferative advantage in mesenchymal lineages like fibroblasts.

overall_design: We first transduced H1 human embryonic stem cells (hESCs) with a library of 51 cancer drivers and injected them into immunodeficient mice to form round 1 teratomas in 4 mice. Cells prior to injection as well as each round 1 teratoma was dissociated and profiled via single cell RNA-seq. Cells from the dissociated round 1 teratomas were reinjected into immunodeficient mice to form round 2 tumors, 2 of which were profiled via single cell RNA-seq.  We then removed the most potent drivers in the initial screen, c-MYC and myristoylated AKT1, and formed a driver sub-library. H1 hESCs were transduced with this sub-library and injected  into immunodeficient mice to form round 1 teratomas. 3 of these round 1 teratomas were profiled via single cell RNA-seq. Cells from the dissociated round 1 teratomas were reinjected into immunodeficient mice to form round 2 tumors, 2 of which were profiled via single cell RNA-seq.  For each of the single cell RNA-seq runs, driver barcodes were associated with cell barcodes by amplifying from unfragmented cDNA.  Individual drivers which were top hits (c-MYC, myr-AKT1, c-MYC + myr-AKT1 and MEK1 (S218D/S222D)) were also profiled via bulk RNA-seq (2 tumors each) and barcode enrichment was assessed from genomic DNA.

first: Daniella

last: McDonald

first: Amir

last: Dailamy

first: Yan

last: Wu

first: Thekla

last: Cordes

first: Kun

last: Zhang

first: Ann

last: Tipps

first: Christian

last: Metallo

first: Prashant

last: Mali


title: Transcriptome profiling of human Natural Killer (NK) cells overexpressing the transcription factors T-BET or EOMES

summary: T-BET and EOMES are key transcription factors in the development of mature NK cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES in human umbilical cord blood-derived hematopoietic progenitor cells (HPC) and cultured them in vitro in an NK cell differentiation model. On day 21 of culture mature stage 4 (CD56+CD94+CD16-) and stage 5 (CD56+CD94+CD16+) NK cells from T-BET or EOMES overexpression and control cultures were sorted, whereafter mRNA was isolated and transcriptome analysis was performed by RNA sequencing. Evaluation of the transcriptome in mature NK cells with T-BET or EOMES overexpression could reveal the molecular mechanisms of how T-BET and EOMES play a role in terminal NK cell maturation.

overall_design: mRNA profile of T-BET and EOMES overexpressing NK cells from day 21 of culture in comparison to control transduced NK cells.


title: Profiling of the chromatin landscape of hematopoietic progenitor cells (HPC) transduced with transcription factors T-BET and EOMES [ATAC-seq HPC]

summary: T-BET and EOMES are key transcription factors in the development of mature Natural Killer (NK) cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES in human umbilical cord blood-derived HPC and cultured them in vitro in an NK cell differentiation model. To evaluate the effect of early overexpression of T-BET and EOMES in HPC, the chromatin landscape was uncovered using assay for transposase-accessible chromatin (ATAC) sequencing. In this way, the regulatory effect of T-BET and EOMES overexpression on the epigenome of HPC was evaluated.

overall_design: Profiling of the chromatin accessibility landscape by Fast-ATAC sequencing of HPC transduced with transcription factors T-BET and EOMES in comparison to control transduced cells.


title: Transcriptome profiling of hematopoietic progenitor cells (HPC) transduced with transcription factors T-BET or EOMES

summary: T-BET and EOMES are key transcription factors in the development of mature Natural Killer (NK) cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES in human umbilical cord blood-derived HPC and cultured them in vitro in an NK cell differentiation model. To evaluate the effect of early overexpression of T-BET and EOMES in HPC, transcriptome profiling was performed on T-BET and EOMES overexpressing HPC and compared to control transduced HPC by RNA sequencing on day 0 of culture.

overall_design: mRNA profile of cord blood-derived HPC transduced with transcription factors T-BET or EOMES in comparison to control transduced cells.


title: mRNA profiling of Trihelix transcription factor ASIL mutants at the late heart stage embryos

status: Public on Aug 26 2021

city: Lancaster

first: Pablo

last: Jenik

state: PA

address: PO Box 3003

department: Department of Biology

institute: Franklin and Marshall College

summary: The current study aimed at exploring the roles of ASIL1 and ASIL2 trihelix transcription factors in repressing the maturation program during early embryogenesis.

overall_design: mRNA profiling was performed on late heart stage embryos in single (asil1-1, asil2-1, asil2-2) and double ASIL mutants (asil1-1 asil2-1, asil1-1 asil2-2), as well as their corresponding wild-type ecotype (Columbia-0 for asil1-1 and asil2-1; Landsberg erecta for asil2-2).  Three biological replicates were collected for each genotype.

first: Julie

last: Pelletier

first: Yuchi

last: Wang

first: John

last: Harada


title: mRNA profiling of Trihelix transcription factor ASIL mutants


title: Expression data from germinal center (GC) B cells of Tnfrsf13B wild type, knockout and A144E mutant mice 10 days post immunization with allogenic cells

first: Mayara

last: Garcia de Mattos Barbosa

state: Michigan

address: 1150 Medical Science Center Drive MSRB1 room A520-1

department: Transplant Surgery

summary:
B cells in the germinal center of the spleen produce antibodies in responses to different antigens. Tnfrsf13b knockout and A144E mice can underlie more aggressive B cells effector responses, possibly at the cost of inadvertent pathogenicity; however, neither the amplitude nor the pathogenicity are simple functions of the amount of antigen-specific IgG produced.
We used microarrays to detail the global program of gene expression of germinal center B cells and identified distinct classes of up-regulated genes in the different strains of mice with Tnfrsf13b wild type, knockout, monoallilic or biallec A144E missense mutation.

overall_design: C57BL/6 background mice with wild type Tnfrsf13b, Tnfrsf13b-KO or harboring monoallelic (A144E-WT) or biallelic (A144E-A144E) Tnfrsf13b A144E mutations were immunized for 10 days via intraperitoneal injection of 50 million BALB/c splenocytes and thymocytes in PBS. Splenocytes were stained  with FVS780, anti-CD19 APC, GL7 PE and CD95 FITC and germinal center B cells were sorted and the RNA was extracted for gene expression analysis via microarray.

first: Marilia

last: Cascalho